Method and device for producing vaccine

ABSTRACT

A method of making a vaccine using animal derived component free (ADCF) cell culture technology, including the steps of attaching ADCF-adapted cells to a microcarrier including an attachment mechanism for attaching filipodia of the cells, the microcarrier being in a culture, growing the cells in ADCF maintenance media, infecting the cells with vaccine media, producing virus within the cells, and harvesting the virus. A vaccine produced by the above method in a pharmaceutically acceptable carrier. A vaccine production structure of ADCF-adapted cells removably attached to microcarrier beads including an attachment mechanism for attaching filipodia of the cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. Section119(e) of U.S. Provisional Patent Application No. 60/772,156, filed Feb.10, 2006, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a method and device for producing avaccine. More specifically, the present invention relates to aprotein-free cell culture process for producing a vaccine.

2. Description of the Related Art

Immunization to protect against communicable disease is one of the mostsuccessful and cost-effective practices of modern medicine. Smallpox hasbeen completely eliminated by vaccination, and the incidence of manyother dreaded diseases such as polio and diphtheria has been drasticallyreduced through immunization programs. However, vaccines, especiallythose based on the use of inactivated viruses, vary in effectiveness.For example, while the currently licensed influenza vaccine isreportedly over 80% efficacious in young adults, it is onlyapproximately 60% efficacious in adults 65 years of age and older, andless than 50% effective in children under 2 years of age. The recentlylicensed chicken pox vaccine is reportedly approximately 70%efficacious, and there are currently no effective vaccines against manyimportant viral diseases including those caused by respiratory syncytialvirus, parainfluenza 3 virus, Rotavirus, and the human immunodeficiencyvirus. In some cases, licensed inactivated viral vaccines may causeadverse reactions that have prevented their use at the higher dosagesneeded to improve efficacy.

Inactivated virus vaccines confer protection by stimulating immuneresponses to proteins found in the free virus. Antibodies to the matureenvelope proteins found on free virus may be optimal in blocking theinitial events of infection (such as virus binding to a cell receptorand attachment and entry into a cell) following exposure to a virus, butmay be sub-optimal once a virus has entered a cell. Once infected, thecells and the cell-associated immature virions contain precursors to themature envelope proteins. These precursor proteins may stimulate moreoptimal immune responses for stemming the spread of infection andpreventing clinical illness when the body's first line of defense,antibodies to free virus, does not completely prevent all viruses frominfecting cells.

Inactivated virus vaccines are typically produced from virus that hasbeen grown in animal cells, e.g. embryonated eggs for influenza, whichare then inactivated by treatment with chemicals such as formalin.Attenuated vaccines for measles and chickenpox are produced by growingweakened virus in cell cultures. Advances in the understanding of thepathogenesis of viral infections and recombinant DNA technology have ledto the identification and production of specific viral proteins for usein subunit viral vaccines. These have been particularly successful inthe formulation of a subunit vaccine against the hepatitis B virus.

Most existing licensed vaccines and vaccines in development, whetherbased on inactivated viruses or recombinant DNA technology, relyprimarily on immune responses to the mature virus, or, in a few examplesof experimental, recombinant DNA-based vaccines, immune responses toantigens found in the cell-associated form of the virus, orvirus-infected cells. Both the killed virus and attenuated virusapproaches on the one hand and the recombinant DNA approaches on theother hand have their advantages and their limitations. While the cellculture and embryonated egg methods are used to grow whole virus veryinexpensively, they are not very efficient methods for the commercialproduction of the viral precursor proteins found in the infected cellsand the cell-associated forms of the virus. This is because thesemethods act like miniature assembly lines and, while a large amount ofmature virus accumulates in the cell cultures or the eggs at any giventime, a much smaller amount of virus is actually in the process of beingassembled. Therefore, the purified virus used to make the vaccinecontains very little, if any, of the envelope precursor or otherprecursor proteins. On the other hand, viral membrane glycoproteins, ineither their mature or precursor form, can be efficiently produced byrecombinant DNA technology. When native conformational structure isneeded to produce functional, neutralizing antibodies, the use ofrecombinant technology employing mammalian cell or insect cellsubstrates is preferred. However, production of viral vaccine proteinsin insect or mammalian cells by recombinant methods is generally moreexpensive on a per milligram protein basis than cell culture and eggproduction methods.

Adverse reactions from vaccines may arise from impurities or frombiologic properties of the vaccine proteins (antigens) responsible forconferring protective immunity. For example, the contaminating eggprotein present in the licensed influenza vaccines may be largelyresponsible for the adverse reactions associated with these products.

Mature viral proteins present in vaccines may have biologic propertiesthat are responsible for adverse reactions. Uptake by mononuclear cellsand granulocytes of inactivated influenza virus mediated by the maturehemagglutinin may also be responsible for adverse reactions. The matureHIV envelope glycoprotein (gp120) in some experimental vaccines againstHIV may bind to the CD4 receptor of T4 lymphocytes and alter normalimmune function. It would be desirable to reduce potential adversereactions in the vaccine preparations.

The viral envelope proteins in inactivated virus vaccines aresubstantially glycosylated. While glycosylation is important inmaintaining conformational structure of these proteins it may alsoreduce their immunogenicity. These proteins in either the mature ofprecursor form can be produced with trimmed carbohydrate residues withrecombinant baculovirus expression vectors and insect cells. Thebaculovirus-produced proteins retain sufficient native conformation tostimulate functional neutralizing antibodies and may provide greaterimmunogenicity than highly glycosylated native proteins.

Infection by influenza virus causes substantial illness and prematuredeath worldwide. Immunization with vaccines comprised of preparations ofinactivated influenza viruses is currently the most useful practice forreducing disease from viral influenza. The vaccines confer protectionagainst infection and disease by stimulating the production of immuneresponses to the hemagglutinin (HA), neuraminidase (NA), nucleoproteins(NP, M1) and possibly other proteins of component strains (Murphy, B.R., et al., N. Engl. J. Med. 268:1329-1332 (1972) and Kendal, A. P., etal., J. Infect. Dis. 136:S415-24 (1986)). The most important of these isthe production of neutralizing antibodies to HA (Ada, G. L., and Jones,P. D., Curr. Top. Microbiol. Immunol. 128:1-54 (1986)). The currentlyavailable inactivated vaccines nevertheless have limitations, includingsub-optimal immunogenicity and efficacy in adults 65 years of age andolder and very young children and under utilization in part due to poorpatient acceptance in connection with the belief that such vaccines arenot very effective and fears of adverse reactions (Nichol, K. L., etal., Arch. Int. Med. 152:106-110 (1992)). The perception of lack ofeffectiveness arises in part from variations in potency from year toyear and the association of many non-influenza respiratory tractillnesses with influenza.

The mature influenza virus contains both HA and NA proteins in its outerenvelope. The HA is present as trimers. Each HA monomer consists of twopolypeptides (HA1 and HA2) linked by a disulfide bond. Thesepolypeptides are derived by cleavage of a single precursor protein, HA0,during maturation of the influenza virus. In part, because thesemolecules are tightly folded, the HA0 and the mature HA1 and HA2 differslightly in their conformation and antigenic characteristics.Furthermore, the HA0 is more stable and resistant to denaturation and toproteolysis. Recently it has been reported that a baculovirus/insectcell culture derived recombinant HA0 conferred protective immunity toinfluenza (Wilkinson, B., Micro Gene Sys Recombinant Influenza Vaccine,PMA/CBER Viral Influenza Meeting, Dec. 8, 1994). One limitation ofrecombinant HA0 vaccines is their inability to stimulate immuneresponses against non-HA antigens that may provide greater and moredurable protection, especially for high-risk populations that do notrespond well to immunization.

Current production of human influenza vaccine takes place primarily infertile chicken eggs. Worldwide several hundred million of eggs are usedeach year to produce vaccine for the influenza season. The currentproduction cycle (beginning with identification of the anticipated virusstrains expected to be present in the forthcoming influenza season) ismany months long. The current production processes that use fertile eggsas tiny bioreactors is labor intensive, expensive and fraught withvariables, such as the seasonal availability and variation of propertiesof the eggs.

It would therefore be desirable to provide improved virus vaccinepreparations that do not exhibit as many of the limitations anddrawbacks observed with the use of currently available vaccines.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of making a vaccine using animalderived component free (ADCF) cell culture technology by attachingADCF-adapted cells to a microcarrier including an attachment mechanismfor attaching filipodia of the cells, the microcarrier being in aculture, growing the cells in ADCF maintenance media, infecting thecells with vaccine media, producing virus within the cells, andharvesting the virus.

The present invention further provides a vaccine produced by the abovemethod in a pharmaceutically acceptable carrier.

The present invention also provides a vaccine production structure ofADCF-adapted cells removably attached to microcarrier beads including anattachment mechanism for attaching filipodia of the cells.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1 is a photograph of synchronized, evenly distributed cellattachment to Hillex 2, the impeller is on until cell attachment isachieved, typically within 30 minutes;

FIG. 2 shows Vero cells spreading (white) on Hillex within 24 hoursduring intermittent stirring;

FIG. 3 shows Vero cells growing (white) on Hillex after 72 hours inculture;

FIG. 4 shows confluent Vero ADCF cells on Hillex after 4 days inculture;

FIG. 5 shows growth curve; x axis hours in culture and y axis number ofcells;

FIG. 6 shows cells properly maintained with ADCF DMEM prior toinfection;

FIG. 7 shows cells improperly maintained with ADCF growth mediaresulting in cells slough off microcarriers; and

FIG. 8 shows a comparison of A/Panama H3N2 harvest titer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of making a vaccine usingmicrocarrier beads. Specifically, the a vaccine is made using animalderived component free (ADCF) cell culture technology by attachingADCF-adapted cells to a microcarrier including an attachment mechanismfor attaching filipodia of the cells, the microcarrier being in aculture, growing the cells in ADCF maintenance media, infecting thecells with vaccine media, producing virus within the cells, andharvesting the virus. This method utilizes microcarrier bead technologyto develop vaccines via the methodology disclosed in the examples. Thevaccines developed are any vaccine that can be produced in the mannerdisclosed herein. Further, the method of the present invention can beused to develop new vaccines. The present invention also provides avaccine production structure of the ADCF-adapted cells which areremovably attached to the microcarrier beads as described above andfurther detailed below.

The present invention utilizes microcarrier beads which have anattachment mechanism of a micro-porous surface. This micro-poroussurface provides stronger attachment of cells to the microcarrier beadthrough the extension of filipodia from the cells. The preferredmicrocarrier bead has a density in the range of 1.04 to 1.1 g/cc.Additional sized beads or beads made of heavier material are suited forcertain applications.

A number of different materials including glass, polystyrene plastic,acrylamide, solid collagen, porous collagen, cellulose and liquidfluorocarbons have been successfully used as microcarriers (Varani, etal. (1983); Nielson, et al. (1980); Obrenovitch, et al. (1982); Giard,et al., (1977); Gebb, et al., (1982)). In addition, microcarriers withone or more adhesive peptides attached to the surface through covalentor noncovalent linkages have been used (Keese, et al. (1983); Varani, etal. (1988); Varani, et al., (1986)). To be useful as a microcarrier, amaterial must have a surface chemistry which supports cell attachmentand growth, and must not be toxic to the cells or to the elaboratedproducts. The ideal microcarrier has a diameter of approximately 75-225although larger or smaller sizes (U.S. Pat. No. 5,114,855 (May 1992); J.Varani, S. Josephs and W. Hillegas, “Human Diploid Fibroblast growth inpolystyrene microcarriers in aggregates”, Cytotechnology, 22: 111-117(1996)) have been used.

Two types of microcarrier products are widely-used in the industrytoday. These are the i) dextran-based microcarriers (Cytodex I,DEAE-dextran; and Cytodex III, porcine collagen-coated dextran) made inSweden and sold by Amersham-Pharmacia Biotech of the United Kingdom andii) the coated polystyrene based microcarriers made in the United Statesby SoloHill. Microcarriers made by SoloHill have been successfullyintegrated into manufacturing processes in the United States, Europe andJapan. SoloHill makes a porcine collagen-coated polystyrene microcarrierbead, which is heavily used in the animal health industry to produceviral vaccines. Smaller amounts of Solohill's glass-coated polystyrenemicrocarriers have also found a use in industry, and an intense interesthas developed in the recently-released ProNectin F*—coated polystyrenebeads, largely because they are free of animal proteins. *ProNectin F isa genetically engineered protein incorporating multiple copies of thecell attachment ligand (RGD) from fibronectin. The microcarrier beadmade with a t-butyl styrene core can be further coated if important tocell culture. This coating can include, but is not limited to porcine,bovine or human collagen, or ProNectin F, a recombinant fibronectin, orother natural or synthetic peptides. This coating is applied in the samemanner as is utilized for standard collagen-coated polystyrenemicrocarriers. (See U.S. Pat. No. 4,944,388 to Hillegas, et al.).

The examples below detail the protocols used for making influenzavaccine using animal derived component free (ADCF) cell culturetechnology. Cell culture technology presents the advantages ofautomation through large batch, computer controlled bioreactors, andthus a highly reproducible manufacturing protocol. The uniqueness ofthis invention is that this process; (a) uses adapted mammalian cellssuch as Vero or Madin-Darby Canine Kidney (MDCK) cells, (b) uses mediaformulations that contain no animal derived components, (c) usesmicrocarriers such as the protein-free types developed, produced andsold by SoloHill and (d) is developed for commercial-scale manufacturingusing computer controlled bioreactors capable of producing batches ofhigh-titer vaccine of 1,000 liters or even larger. An added advantage tothis cell culture process compared to the conventional egg-basedtechnology is the rapid turnaround time when an influenza pandemicarises. Thus the described cell-culture based technology is vastly morecapable of producing appropriate vaccine to counteract an outbreak of amonovalent virus.

Vero cells, adapted to animal derived component free (ADCF), Hillex 2microcarrier culture conditions infected with various strains ofinfluenza virus generate high virus yields and exemplify the ADCFupstream process described in this application. Critical stepsidentified for the ADCF process for influenza production include 1)robust ADCF-adapted cell line, 2) removal of cells from substrate usingADCF trypsin resulting in a single cell suspension for cultureinitiation, 3) uniform cell attachment to Hillex 2 microcarrier, 4)timely cell spreading to Hillex 2 microcarrier following attachment, 5)cell growth as a function of ADCF media management, 6) maintenance ofrobust cells prior to influenza infection using ADCF maintenance media,7) Vero cell adapted influenza virus working seeds with adequate titersto infect the cell cultures, and 8) timing of cell infection relative tocell seeding density on Hillex 2 microcarrier and growth phase of thecells where all factors synergistically produce high virus yields.

To complete the manufacturing process, following the upstream process asdescribed, the influenza strains that serve as antigens may undergoextensive down stream processing before the vaccine is formulated intothe final product. Downstream processing is not within the scope of thisapplication.

Previously, for influenza vaccine production, the Vero cell line isassociated with unsatisfactory virus yields and numerous technicaldifficulties. Although multiple replication cycles of influenza strainsare possible in Vero cell cultures with repeated doses ofL-I-tosylamide-2-phenylethyl chloromethyl ketone-treated (TPCK) orTrypZean (a ADCF bovine trypsin recombinant expressed in corn,Sigma-Aldrich Co.) virus yields of most strains in Vero cells are stilltoo low for cost effective vaccine production. Applicants demonstrate,however, that limited passage in Vero cells of wild-type candidates athigh multiplicity of infection produce titers satisfactory for virusseed and subsequent virus production eliminating the need for extensiveand elaborate procedures like reassortment or reverse genetics.

Historically, ADCF adapted cells have been difficult to synchronize inan ADCF microcarrier culture system. First, ADCF cells are difficult toattach to ADCF microcarriers, then cell distribution tends to be skewedleading to numerous production problems, and finally cells fail tospread eventually falling off the microcarriers. Gelatin-coatedmicrocarriers, therefore, remain a component in the so called ADCFculture platform. Applicants have demonstrated synchronized cellattachment, consistent cell distribution among microcarriers, a goodrate of cell spreading, and good cell growth as required for asatisfactory virus infection using ADCF Hillex 2 microcarriers.

Prior to culture initiation, equipment and materials are prepared.Culture vessels can include spinner flasks equipped with impellers withstir plates controlling the speed of the impeller or computer controlledbioreactors equipped with set points for temperature, dissolved oxygen,pH controlled by CO₂, acids or bases, impeller rpm ranging from 0 to 60,Culture vessels are configured before sterilizing for cultureinitiation. Hillex 2 or other ADCF microcarriers are sterilized in DIwater or DPBS. Media without phenol red is recommended, but notrequired, for Hillex 2.

ADCF adapted Vero cells are expanded from a working cell bank by serialpassages in T-flasks, roller bottles, microcarrier cultures or variousother platforms until the desired cell counts are achieved for batchproduction of each of the various strains of influenza virus.

Prior to culture initiation, confluent ADCF adapted cells are removedfrom the substrate using ADCF-type trypsin forming a single cellsuspension which is transferred to the ADCF Hillex 2 culture system.

To remove cells from the substrate, monolayers are rinsed with DPBStwice and or a weak solution of EDTA in DPBS with 10-30 minuteincubation periods before cells are dissociated from the substrate usingADCF cell detachment solution such as HYQtase (Hyclone Laboratories,Inc.) or TrypLE select (Invitrogen Corporation). A satisfactory processstep results in a viable, single cell suspension essential formicrocarrier culture initiation.

A successful Hillex 2 culture initiation is dependent upon 1) singlecell suspension with 92% viability or better, 2) pH management, 3) rpmset at 50-60, 4) culture vessel fitted with a proper impeller to keepthe Hillex 2 microcarriers in suspension, 5) optimized temperature rangefor optimized cell growth, and 6) dissolved oxygen of 5% or higher.

Once the desired cell density is achieved with ADCF media, the cellsmonolayered on ADCF Hillex 2 microcarriers must be properly maintainedfor a successful influenza infection process step. The ADCF culture atthis stage in the upstream process must be robust to survive therepeated doses of enzyme used to cleave the HA protein necessary formultiple rounds of virus replication in Vero cell cultures. Since thecurrently available ADCF media are designed explicitly for growth, notmaintenance, required media exchanges with ADCF media results inovergrowth causing the cell monolayers to slough off the microcarriers.Applicants demonstrate that maintaining robust cells, not with ADCFgrowth media, rather with a maintenance-type media, ADCF DMEM withoutadditives except L-glutamate for example, properly maintains confluent,robust cells at a substantially reduced cost for the subsequentinfluenza virus infection step leading to high virus titers.

Soon after the cells are 85% confluent to barely contiguous, the ADCFmedia is replaced with a maintenance media without animal proteins likeDMEM supplemented with only L-glutamine. The cells will remain robust inthe later exponential phase until the infection step is initiatedtypically within 2 to 24 hours.

The method of the present invention can be used for wild type influenzavirus adaptation to Vero cells and the ADCF upstream process conditionsmade possible by the unique ADCF properties of Hillex 2 supporting cellattachment, cell spreading, cell growth, robust cell maintenance duringVero cell infection using wild-strain influenza seed virus adapted toVero cells.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided for thepurpose of illustration only, and are not intended to be limiting unlessotherwise specified. Thus, the invention should in no way be construedas being limited to the following examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

Examples Methods:

General methods in molecular biology: Standard molecular biologytechniques known in the art and not specifically described are generallyfollowed as in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York (1989), and in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989) and in Perbal, A Practical Guide to MolecularCloning, John Wiley & Sons, New York (1988), and in Watson et al.,Recombinant DNA, Scientific American Books, New York and in Birren et al(eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold SpringHarbor Laboratory Press, New York (1998) and methodology as set forth inU.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057and incorporated herein by reference. Polymerase chain reaction (PCR)was carried out generally as in PCR Protocols: A Guide To Methods AndApplications, Academic Press, San Diego, Calif. (1990). In-situ(In-cell) PCR in combination with Flow Cytometry can be used fordetection of cells containing specific DNA and mRNA sequences (Testoniet al; 1996, Blood 87:3822.)

General methods in immunology: Standard methods in immunology known inthe art and not specifically described are generally followed as inStites et al.(eds), Basic and Clinical Immunology (8th Edition),Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi (eds),Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York(1980).

Antibody Production

Antibody Production: Antibodies may be either monoclonal, polyclonal, orrecombinant. Conveniently, the antibodies may be prepared against theimmunogen or portion thereof. For example, a synthetic peptide based onthe sequence, or prepared recombinantly by cloning techniques or thenatural gene product and/or portions thereof may be isolated and used asthe immunogen. Immunogens can be used to produce antibodies by standardantibody production technology well known to those skilled in the art asdescribed generally in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988 andBorrebaeck, Antibody Engineering—A Practical Guide, W.H. Freeman andCo., 1992. Antibody fragments also can be prepared from the antibodiesand include Fab, F(ab′)₂, and Fv by methods known to those skilled inthe art.

For producing polyclonal antibodies, a host, such as a rabbit or goat,is immunized with the immunogen or immunogen fragment, generally with anadjuvant, and if necessary, coupled to a carrier; antibodies to theimmunogen are collected from the sera. Further, the polyclonal antibodycan be absorbed such that it is monospecific. That is, the sera can beabsorbed against related immunogens so that no cross-reactive antibodiesremain in the sera, rendering it monospecific.

For producing monoclonal antibodies, the technique involveshyperimmunization of an appropriate donor with the immunogen, generallya mouse, and isolation of splenic antibody producing cells. These cellsare fused to a cell having immortality, such as a myeloma cell, toprovide a fused cell hybrid that has immortality and secretes therequired antibody. The cells are then cultured, in bulk, and themonoclonal antibodies harvested from the culture media for use.

For producing recombinant antibody (see generally Huston et al, 1991;Johnson and Bird, 1991; Mernaugh and Mernaugh, 1995), messenger RNAsfrom antibody producing B-lymphocytes of animals, or hybridoma, arereverse-transcribed to obtain complimentary DNAs (cDNAs). Antibody cDNA,which can be full or partial length, is amplified and cloned into aphage or a plasmid. The cDNA can be a partial length of heavy and lightchain cDNA, separated or connected by a linker. The antibody, orantibody fragment, is expressed using a suitable expression system toobtain recombinant antibody. Antibody cDNA can also be obtained byscreening pertinent expression libraries.

The antibody can be bound to a solid support substrate or conjugatedwith a detectable moiety or be both bound and conjugated as is wellknown in the art. (For a general discussion of conjugation offluorescent or enzymatic moieties see Johnstone & Thorpe,Immunochemistry in Practice, Blackwell Scientific Publications, Oxford,1982). The binding of antibodies to a solid support substrate is alsowell known in the art. (For a general discussion, see Harlow & LaneAntibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPublications, New York, 1988 and Borrebaeck, Antibody Engineering—APractical Guide, W.H. Freeman and Co., 1992) The detectable moietiescontemplated with the present invention can include, but are not limitedto, fluorescent, metallic, enzymatic and radioactive markers such asbiotin, gold, ferritin, alkaline phosphatase, b-galactosidase,peroxidase, urease, fluorescein, rhodamine, tritium, ¹⁴C and iodination.

Delivery of Gene Products/Therapeutics (Compound):

The compound of the present invention is administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In the method of the present invention, the compound made by the stepsof the present invention can be administered in various ways. It shouldbe noted that it can be administered as the compound or aspharmaceutically acceptable salt and can be administered alone or as anactive ingredient in combination with pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles. The compounds can beadministered orally, subcutaneously or parenterally includingintravenous, intraarterial, intramuscular, intraperitoneally, andintranasal administration as well as intrathecal and infusiontechniques. Implants of the compounds are also useful. The patient beingtreated is a warm-blooded animal and, in particular, mammals includingman. The pharmaceutically acceptable carriers, diluents, adjuvants andvehicles as well as implant carriers generally refer to inert, non-toxicsolid or liquid fillers, diluents or encapsulating material not reactingwith the active ingredients of the invention.

It is noted that humans are treated generally longer than the mice orother experimental animals exemplified herein which treatment has alength proportional to the length of the disease process and drugeffectiveness. The doses may be single doses or multiple doses over aperiod of several days, but single doses are preferred.

The doses may be single doses or multiple doses over a period of severaldays. The treatment generally has a length proportional to the length ofthe disease process and drug effectiveness and the patient species beingtreated.

When administering the compound of the present invention parenterally,it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.According to the present invention, however, any vehicle, diluent, oradditive used would have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various other ingredients, asdesired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, adjuvants, additives, anddiluents; or the compounds utilized in the present invention can beadministered parenterally to the patient in the form of slow-releasesubcutaneous implants or targeted delivery systems such as monoclonalantibodies, vectored delivery, iontophoretic, polymer matrices,liposomes, and microspheres. Examples of delivery systems useful in thepresent invention include: U.S. Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

A pharmacological formulation of the compound utilized in the presentinvention can be administered orally to the patient. Conventionalmethods such as administering the compounds in tablets, suspensions,solutions, emulsions, capsules, powders, syrups and the like are usable.Known techniques that deliver it orally or intravenously and retain thebiological activity are preferred.

In one embodiment, the compound of the present invention can beadministered initially by intravenous injection to bring blood levels toa suitable level. The patient's levels are then maintained by an oraldosage form, although other forms of administration, dependent upon thepatient's condition and as indicated above, can be used. The quantity tobe administered can vary for the patient being treated and will varyfrom about 100 ng/kg of body weight to 100 mg/kg of body weight per dayand preferably can be from 1 mg/kg of body weight to 10 mg/kg of bodyweight per day.

Example 1

To support the Vero-ADCF microcarrier process, satisfactory Vero celladapted master virus seeds must be prepared.

For conventional, large scale influenza virus production, strains ofinfluenza selected for annual vaccine production are likely configuredfor seed virus by reassorting the wild-type strain with A/PuertoRico/8/34 (PR8), a master strain adapted to grow in embryonated eggs forenhanced virus yields. Re-assorted viruses are screened for surfaceantigen, hemagglutinin (HA) and neuraminidase (NA), genes from targetedwild-type strains and structural genes from the PR8 master strain virus.These reassorted influenza viruses are then grown in embryonated eggs toexpand viruses used as working seed for production batches.

Preparing Influenza virus seed for the Vero ADCF platform typicallyinvolves adaptation to Vero cells to increase titers suitable for masterseed.

Govorkova, et. al. reported A/England/1/53 titers of 8.37 log₁₀TCID₅₀/ml only after 20 passages using a low MOI in Vero cells.Considering the time constrains, this protocol is not practical forcommercial-scale manufacturing. However, there is demonstrated apractical approach to developing virus seed for influenza production.Five of six recent wild-type influenza virus strains isolated andexpanded in embryonated eggs did indeed require adaptation to Vero cellsbefore they were regarded as suitable for the seed virus used to produceconsistently high titers. However, only five or less passages wererequired for these five strains of human influenza virus grown in eggsand serial passaged at a high MOI (1 to 10) in Vero cells over a periodof only 12 days; A/Beijing/262/95, A/Moscow/10/99, A/Sydney/05/97,B/Harbin/07/94 and A/New Caledonia/20/99. Only A/Panama/2007/99 requiredno multiple passage adaptation. These results indicate the adaptabilityof human influenza virus strains to Vero cells and the suitability ofthis method for preparing influenza master seeds used for seasonalmanufacturing of influenza virus antigens. Future studies are requiredto determine the genetic stability of human influenza virus strainsusing this protocol.

Influenza Virus Adaptation to Vero Cells Log₁₀ TCID₅₀/ml Strain Egg Verop.1 Vero p.4 A/Beijing 8.5 0 7.6 A/New Caledonia 8.2 0 8.1 A/Moscow 7.54.8 7.8 A/Sydney 8.0 6.3 7.5 A/Panama 7.8 7.9 8.0 B/Harbin 7.2 0 7.4

Prior to culture initiation, equipment and materials are prepared:Culture vessels can include spinner flasks equipped with impellers withstir plates controlling the speed of the impeller or computer controlledbioreactors equipped with set points for temperature, dissolved oxygen,pH controlled by CO₂, acids or bases, impeller rpm ranging from 0 to 60,Culture vessels are configured before sterilizing for cultureinitiation. Hillex 2 or other ADCF microcarriers are sterilized in DIwater or DPBS. Media without phenol red is recommended, but notrequired, for Hillex 2.

ADCF adapted Vero cells are expanded from a working cell bank by serialpassages in T-flasks, roller bottles, microcarrier cultures or variousother platforms until the desired cell counts are achieved for batchproduction of each of the various strains of influenza virus.

Prior to culture initiation, confluent ADCF adapted cells are removedfrom the substrate using ADCF-type trypsin forming a single cellsuspension which is transferred to the ADCF Hillex 2 culture system.

To remove cells from the substrate, monolayers are rinsed with DPBStwice and or a weak solution of EDTA in DPBS with 10-30 minuteincubation periods before cells are dissociated from the substrate usingADCF cell detachment solution such as HYQtase (Hyclone Laboratories,Inc.) or TrypLE select (Invitrogen Corporation). A satisfactory processstep results in a viable, single cell suspension essential formicrocarrier culture initiation.

A successful Hillex 2 microcarrier culture initiation is dependentupon 1) single cell suspension with 92% viability or better, 2) pHmanagement, 3) rpm set at 50-60, 4) culture vessel fitted with a properimpeller to keep the. Hillex 2 microcarriers in suspension, 5) optimizedtemperature range for cell growth data, and 6) dissolved oxygen of 5% orhigher. For example, Hillex 2 microcarriers are transferred to theculture vessel in ½ to ¾ volume of the total culture volume at least 1hour before cell suspension is added—1300 cm² to 2600 cm² or greatersurface area per 200 mls of ADCF media. Single cell suspension istransferred to the culture vessel with the impeller running at a rate of50-60 rpm, the pH at 7.5-7.6, and temperature at 37° C. (Seeding densityis 10 to 20 cells per microcarrier added to the culture depending onmany factors including manufacturing schedule.) For synchronized, evenlydistributed cell attachment to Hillex 2, the impeller is on until cellattachment is achieved, typically within 30 minutes as shown in FIG. 1.

Conditions for Cell Spreading on Hillex 2 Microcarriers.

Once 90% of the cells have attached to the microcarriers, cultureconditions for the cell attachment phase are imposed. Set point for pHcan be adjusted to 7.2-7.4. Intermittent stirring cycle is initiated fora period of up to 24 hours. For example—3 minutes on at 50-60 rpm and 30minutes off. The current experience is for 3 hours to 24 hours dependingon the schedule and, indeed, the rate of spreading which is based on thecondition of the cells at the time the culture was initiated. (Ref. cellcontrol)

Once the cells have spread, the impeller is turned on at a constantspeed of 50-60 rpm, for example. (rpm is dependent on the configurationof the impeller assembly within the stir vessel and is determinedempirically.) FIG. 2 shows Vero cells spreading (white) on Hillex within24 hours during intermittent stirring. FIG. 3 shows Vero cells growing(white) on Hillex after 72 hours in culture.

Conditions for ADCF Vero Cell Growth on Hillex 2 Microcarriers.

(24 hrs) pH=7.2 to 7.5. rpm=50-60. d0=10 to 30%. Temperature=37C. Addmedia to final desired culture volume. Exchange media as required basedon empirical growth curve data including the concentration of cellsgrowing on microcarriers per volume of media. For example—½ mediaexchange on day 3 and on day 4 before the cells are confluent on theHillex 2. FIG. 4 shows confluent Vero ADCF cells on Hillex after 4 daysin culture. Insert Vero ADCF confluent 1 picture. FIG. 5 shows growthcurve; x axis number of cells and y axis hours in culture.

Conditions for ADCF Vero Cell Maintenance

Once the desired cell density (cells/cm2) is achieved with ADCF media,the cells monolayered on ADCF Hillex 2 microcarriers must be properlymaintained for a successful influenza infection process step. The ADCFculture at this stage in the upstream process must be robust to survivethe repeated doses of enzyme used to cleave the HA protein necessary formultiple rounds of virus replication in Vero cell cultures. Since thecurrently available ADCF media are designed explicitly for growth, notmaintenance, required media exchanges with ADCF media results inovergrowth causing the cell monolayers to slough off the microcarriers.

Soon after the cells have reached confluence, the ADCF media is replacedwith a maintenance media without animal proteins like DMEM supplementedwith perhaps only L-glutamine. The cells will remain robust in the lateexponential phase until the infection step is initiated typically within12 to 24 hours.

FIG. 6 shows cells properly maintained with ADCF DMEM prior toinfection. Insert Vero ADCF maintained DMEM 020 or 013.

FIG. 7 shows cells improperly maintained with ADCF growth mediaresulting in cells slough off microcarriers.

Conditions for ADCF Vero Cell Influenza Infection

Cell density (cells/cm2) is an important function of influenza cellinfection efficiency and yields. Cells on the microcarriers are rinsedby exchanging the media with maintenance media. Depending on theinfluenza virus two rinses may be required based on influenza straingrowth curve data. Thoroughly rinsing is required to eliminate residualprotein in the culture that tends to inactivate the trypsin which isrequired for proper infection. For the infection, fresh maintenancemedia is added supplemented with L-glutamate ½ to one full volume.Master virus seed is added at a moi of 0.01 to 0.0001 depending ondevelopment results of each strain of influenza. ADCF trypsin is addedimmediately after the seed virus at the proper concentration to cleavethe HA protein. pH=7.4 to 7.6, Temperature =depends on strain, d0=5% to30%, rpm=50-60.

Harvest

Time of harvest depends on type of antigen desired; high viability virusor total antigen including viable and non viable virus. Supernatant isharvested through a biofilter leaving the microcarriers in the vessel orsimply by allowing microcarriers to settle before transferringvirus-laden fluids to a harvest vessel.

A Comparison of A/Panama H3N2 Harvest Titer FIG. 8 shows the followinginformation:

Embryonated eggs log₁₀7.8 TDIC/ml (conventional) Vero serum containingcultures 7.9 (Hillex spinner cultures) Vero ADCF system 8.0 (Hillexspinner cultures)

Throughout this application, author and year, and patents, by number,reference various publications, including United States patents. Fullcitations for the publications are listed below. The disclosures ofthese publications and patents in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology that has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the described invention, theinvention may be practiced otherwise than as specifically described.

REFERENCES

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U.S. PATENT DOCUMENTS

-   U.S. Pat. No. 6,214,618 B1 April 2001 Hillegas et al.

1-33. (canceled)
 34. A method of making an influenza vaccine includingthe steps of: selecting a cell line from the group consisting ofinfluenza-permissive cells having attachment filipodia; attaching saidcells to a plurality of microcarriers including attachment means forattaching said filipodia of the cells; placing said plurality ofmicrocarriers with said attached cells in a culture; suspending saidplurality of microcarriers by an impeller at a speed sufficient tomaintain said microcarriers in a substantially homogenous suspension;growing said cells in a maintenance media; infecting said cells withinfluenza vaccine media; producing influenza virus within said cells;and harvesting said influenza virus.
 35. The method of claim 34 whereinthe attaching step is further defined as removing cells from a substrateusing a protease to result in a single cell suspension, adding saidsingle cell suspension to said culture, uniformly attaching said cellsto said microcarriers, and spreading said cells to said microcarriers.36. The method of claim 34, wherein the producing step is furtherdefined as timing cell infection relative to cell seeding density onsaid microcarriers and growth phase of the cell to synergisticallyproduce high virus yield.
 37. The method of claim 34, wherein theharvesting step is further defined as harvesting supernatant to collectsaid virus.
 38. The method of claim 35, further including the step oftransferring said microcarriers to said culture at least one hour beforeadding said single cell suspension to said culture.
 39. The method ofclaim 34, wherein each of said microcarriers has a micro-porous surface.40. The method of claim 39, wherein said microcarriers have a density of1.04 to 1.1 g/cc.
 41. The method of claim 39, wherein each of saidmicrocarriers has a diameter of 75 to 225 micrometers.
 42. The method ofclaim 34, wherein said microcarriers are composed of a material selectedfrom the group consisting of glass, polystyrene plastic, acrylamide,dextran, solid collagen, porous collagen, porcine collagen, cellulose,and liquid fluorocarbon.
 43. The method of claim 34, wherein saidmicrocarriers are composed of a material chosen from the groupconsisting of porcine collagen coated polystyrene and glass-coatedpolystyrene.
 44. The method of claim 34, wherein said microcarriersinclude at least one adhesive peptide attached to a surface of saidmicrocarriers through covalent or non-covalent linkages.
 45. The methodof claim 34, wherein said microcarriers include a coating chosen fromthe group consisting of porcine collagen, bovine collagen, humancollagen, ProNectin®, recombinant fibronectin, and any other suitablenatural or synthetic peptide.